Control of Colonic Motility in Health and Disease

描述(申请人提供)：结肠粘膜的致病性炎症伴随着环状肌肉收缩的抑制，这对腹泻的症状有重要作用。先前的研究已经证实，收缩能力的抑制是由于介导兴奋-收缩偶联的细胞信号分子表达的特定变化造成的。特别是，减少钙内流的L型钙通道成孔α1c亚单位的表达减少是这一效应的关键因素。细胞信号分子表达的改变是由炎症反应介质介导的，如外肌免疫细胞释放的肿瘤坏死因子α。我们的假设是，肿瘤坏死因子α通过激活转录因子NF-kappaB下调L型钙通道α1c亚单位的基因表达，并通过调节α1c基因的稳定性来抑制收缩。这一假设将在对人结肠环状平滑肌细胞(HCCSMC)进行原代培养的研究中得到验证。该提案的具体目的是调查：1)HCCSMC肌肉中核因子-kappaB的激活是否下调L型钙通道α1c亚单位的表达；2)核因子-kappaB的p50和p65亚基下调α1c表达的分子机制；3)哪些信号分子调节hcCSMC中核因子-kappaB亚单位的激活以响应TNFpha；4)TNFpha是否改变A1c基因的稳定性，如果是，这种影响是否由丝裂原激活的蛋白激酶(MAPK)介导；以及5)环状肌条的收缩是否抑制其收缩能力，如果是，是否这种作用是由p50、p65、PKcheeta和MAPKs介导的吗？这些实验将利用免费的药理学和分子方法。这些发现将为治疗干预提供新的细胞靶点，以调节生理和病理生理学上重要的结肠平滑肌收缩。抑制核因子-kappaB的激活是治疗炎症性疾病的一种新兴疗法。

DESCRIPTION (provided by applicant): Pathogenic inflammation of the colonic mucosa is accompanied by suppression of circular muscle contractility, which makes important contributions to the symptom of diarrhea. Previous studies have established that the suppression of contractility results from specific changes in the expression of cell signaling molecules that mediate excitation-contraction coupling. In particular, a decrease in the expression of the pore-forming alpha1c subunit of L-type calcium channels, which reduces calcium influx, is a key factor in this effect. The alterations in the expression of cell signaling molecules are mediated by inflammatory response mediators, such as TNFalpha released from the immunocytes in the muscularis externa. Our hypothesis is that TNFalpha suppresses contractility by down-regulating the gene expression of the alpha1c subunit of L-type calcium channels through the activation of transcription factor NF-kappaB and by modulating alpha1c mRNA stability. This hypothesis will be tested in studies performed on primary cultures of human colonic circular smooth muscle cells (HCCSMC). The specific aims of the proposal are to investigate: 1) whether NF-kappaB activation in HCCSMC muscle strips down-regulates the expression of alpha1c subunit of L-type calcium channels, 2) the molecular mechanisms of down-regulation of (alpha1c by p50 and p65 subunits of NF-KappaB in response to TNFalpha, 3) which signaling molecules regulate the activation of NF-kappaB subunits in HCCSMC in response to TNFalpha, 4) whether TNFalpha alters the stability of a1c mRNA and, if so, is this effect mediated by mitogen-activated protein kinases (MAPKs), and 5) whether TNFalpha treatment of circular muscle strips suppresses their contractility and, if so, is this effect mediated by p50, p65, PKCzeta and MAPKs. The experiments will utilize complimentary pharmacologic and molecular approaches. The findings will suggest new cellular targets for therapeutic interventions to modulate physiologically and pathophysiologically important contractions of colonic smooth muscle. The inhibition of NF-kappaB activation is an emerging therapy in treating inflammatory disorders.